The goal of this proposal is to define the nature of the kinetochore-DNA interface and determine how centromere-specific chromatin directs the assembly of a functional kinetochore structure in human cells. During each cell division, the entire complement of genetic material must be accurately partitioned to the daughter cells. Even a single chromosome mis-segregation event can be catastrophic, resulting in the loss or gain of hundreds of genes, with severe consequences for development and disease, including tumorigenesis. The central player in directing chromosome segregation is the kinetochore, a large macromolecular structure that mediates attachments between spindle microtubules and a region of each chromosome termed the centromere. Determining the molecular basis for kinetochore function is crucial to understand the defective processes that can give rise to tumor cells, and to evaluate the best targets for the diagnosis and treatment of disease. In vertebrates, centromeres are specified by sequence-independent epigenetic mechanisms that involve the targeted deposition of nucleosomes containing the histone H3-variant, CENP-A. A fundamental unanswered question is how the remainder of the kinetochore is assembled downstream of CENP-A. Our previous work demonstrated that CENP-A is not sufficient to direct assembly of a complete, functional kinetochore structure in human cells. We have identified a heterotetrameric complex comprised of the histone fold proteins CENP-T, -W, -S, and -X as a critical additional component of the kinetochore-DNA interface. The CENP-T-W-S-X complex displays structural similarity to a nucleosome and possesses sequence-independent DNA binding activity. In addition, CENP-T interacts directly with outer kinetochore microtubule-binding proteins to direct kinetochore assembly. Thus, the CENP-T-W-S-X complex plays a pivotal role in connecting the DNA and microtubule interfaces at kinetochores. However, it remains unknown how the CENP-T-W-S-X complex is targeted exclusively to centromeres to generate a single, functional microtubule attachment site on each chromosome. This proposed work will define the mechanisms that direct the centromere localization of the CENP-T-W-S-X complex by assessing: 1) The intrinsic sequence features of the CENP-T-W-S-X complex that are required for its localization, 2) The extrinsic factors that associate with the CENP-T-W-S-X complex to deposit or maintain it at centromeres, and 3) The regulatory modifications and chromatin features that control CENP-T-W-S-X complex localization.